多肽合成工艺流程(英文版).ppt
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peptides (available for fragment condensation).
ClTrt resin prevents the diketopiperazine formation ! Attachment of Cys and His derivatives to the resin is free from
1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH + 8 mmol DIEA in 3-5 mL DCM, for 1.5 h then 0.8 mL MeOH to block the unreacted groups washing with DCM, iPrOH, MeOH, ether
Outline
Resins; Protecting groups; Synthetic protocol; Monitoring; Cleavage technics; Side reactions;
Байду номын сангаас
Fmoc/tBu:
Fmoc
tert-butyl
H3C
H3C H3C C CH3
in the presence of DMAP: Fmoc-Cys(Trt)-OH > Fmoc-Cys(Acm)-OH
However, Fmoc-Cys(Acm) at the C-terminal resultes in side reaction:
NH CH C O CH2
O CH2
P
Acm-S CH2 O
H2N CH
P
Nle is a reference for quantitation.
4-methylbenzhydrylamine-PS-DVB
Pegylated resins: composition of polyethylene glycol (Mw:3000-4000)
and low-cross linker polystyrene gel-type resins.
There are many different resins and most of them are used for special cases and in individual laboratories. Here only the most widely applied resins will be presented. Resins are based on PS-DVB (1%) copolymer.
Applied side chain protecting groups in Fmoc-chemistry
Side chain functional group protecting group name (abbreviation)
-OH (Ser, Thr, Tyr)
CH3 H3C C
tert-butyl (tBu)
NH2(mmol/g) = [A301.V(ml)/e301.m(mg)].106
2. ca. 4-6 mg Fmoc-Aaa-resin +400 mL 50% piperidine/DMF 30 min at RT, then filtration dilute with MeOH to 25 mL
ca. 2 mg Fmoc-Gly-OH +400 mL 50% piperidine/DMF 30 min at RT dilute with MeOH to 25 mL
Capacity of the resin (mmol/g) =
1000.mgly.A3r0es1 in Mgly.mresin.A3g0ly1
SASRIN (Super Acid Sensitive ResIN) (2-methoxy-4-alkoxybenzyl-alcohol resin)
HO CH2
O CH2
P
CH3O
Peptide is cleavable with 0.5-1.0% TFA in DCM resulted in protected peptide fragments.
O
TFA
H3C C CH3
R
O
C
CO
C H
..
NH
piperidine
O
H2C
H2C
CH2 O C NH CH C NH CH C O CH2
O
O
O CH2
Wang-resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin
Type of resins for Fmoc-chemistry
Advantages:
excellent pressure stability (continuous flow synthesis) excellent swelling properties (also in water) high diffusion rates available with many types of functional groups low capacity (0.2-0.6 mmol/g), suitable for the synthesis of aggregating peptides, for on resin cyclisation and fragment condansation.
Piperidine/DMF (1:4, V/V) is added to the mark;
The mixture is shaken thoroughly and left for 25-30 min;
The resin is filtered off and the absorbance of the filtrate is measured at 301 nm (e = 7800).
AcCl or SOCl2 in toluene. Similar to ClTrt resin.
OCH3 H3CO
H2N CH
2,4-dimethoxy-benzhydryl linker NovaSyn R TGR resin
Similar to Rink Amide MBHA resin O
OCH2 C OH
OCH2-P
Rink Amide-AM and Rink Amide-MBHA resins:
OCH3
H3CO Fmoc-HN CH
OCH2-CO-Nle-R
H2N CH2
P
Aminomethyl-PS-DVB
CH3
Peptide cleavage with 90-95% TFA solution.
enantiomerisation!
Determination of loading
(Calculation of the resin capacity) 1.
10-20 mg of dried resin are weighted exactly into a 100 mL measuring flask (for a load of ca. 0.5 meq/g 20 mg is sufficient);
90% TFA solution for effective removal.
-SH (Cys)
trityl (Trt)
For selective deprotection
CH2 NH C CH3 O
acetamidomethyl (Acm)
Racemisation during the attachment of Cys derivatives to the resins
4-Alkoxybenzyl alcohol (Wang) resin:
HO CH2
O CH2
P
Attachment of the first amino acid: Fmoc-Aaa(X)-OH:DIC:DMAP (2:2:0.2 equiv to the resin OH content) in DMF, 1h at RT.
C H3 Trt group can be used if on-resin derivatization is required (glycosylation,
phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs
The final cleavage results in peptides with COOH group at the C-terminus
The resin is not available for the synthesis of peptides with a sequence on the C-terminal that is sensitive for diketopiperazine formation !
The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG) NH2 PEG
HO C H2
O O CH2 C OH
4-hydroxymethylphenoxyacetic acid linker
NovaSyn R TGA resin
Mgly =297
Rink Amide Resin: synthesis of peptides with CONH2 C-terminus
Cleavage with high concentration of TFA can lead to the break down of
OCH3
the linker
byproducts.
Use low TFA concentration and/or
trialkylsilanes in the cleavage mixture.
Peptide-resin bond can be detached
H3CO
with 5% TFA. Removal of protecting Fmoc-HN CH groups needs a separate step.
4-Hydroxymethylphenoxyacetic acid (HMPA) linker:
HO CH2
O CH2 COOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with TFA
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:
Solid phase peptide synthesis
Part II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös L. University Budapest, Hungary
Similar to Wang resin
4-carboxytrityl linker
O
NovaSyn R TGT alcohol resin
HO
C OH Before use the resin must be converted
to the chloride form by heating with
Cl Cl
The final cleavage results in peptides with COOH group at the C-terminus
Cleavage with 90-95% TFA + scavangers results in free peptides Cleavage with AcOH:MeOH(TFE):DCM (1:1:8 or 2:2:6) results in protected
HO CH2 CH3O
O (CH2)3 C OOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (ClTrt) resin:
P
Attachment of the first amino acid:
ClTrt resin prevents the diketopiperazine formation ! Attachment of Cys and His derivatives to the resin is free from
1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH + 8 mmol DIEA in 3-5 mL DCM, for 1.5 h then 0.8 mL MeOH to block the unreacted groups washing with DCM, iPrOH, MeOH, ether
Outline
Resins; Protecting groups; Synthetic protocol; Monitoring; Cleavage technics; Side reactions;
Байду номын сангаас
Fmoc/tBu:
Fmoc
tert-butyl
H3C
H3C H3C C CH3
in the presence of DMAP: Fmoc-Cys(Trt)-OH > Fmoc-Cys(Acm)-OH
However, Fmoc-Cys(Acm) at the C-terminal resultes in side reaction:
NH CH C O CH2
O CH2
P
Acm-S CH2 O
H2N CH
P
Nle is a reference for quantitation.
4-methylbenzhydrylamine-PS-DVB
Pegylated resins: composition of polyethylene glycol (Mw:3000-4000)
and low-cross linker polystyrene gel-type resins.
There are many different resins and most of them are used for special cases and in individual laboratories. Here only the most widely applied resins will be presented. Resins are based on PS-DVB (1%) copolymer.
Applied side chain protecting groups in Fmoc-chemistry
Side chain functional group protecting group name (abbreviation)
-OH (Ser, Thr, Tyr)
CH3 H3C C
tert-butyl (tBu)
NH2(mmol/g) = [A301.V(ml)/e301.m(mg)].106
2. ca. 4-6 mg Fmoc-Aaa-resin +400 mL 50% piperidine/DMF 30 min at RT, then filtration dilute with MeOH to 25 mL
ca. 2 mg Fmoc-Gly-OH +400 mL 50% piperidine/DMF 30 min at RT dilute with MeOH to 25 mL
Capacity of the resin (mmol/g) =
1000.mgly.A3r0es1 in Mgly.mresin.A3g0ly1
SASRIN (Super Acid Sensitive ResIN) (2-methoxy-4-alkoxybenzyl-alcohol resin)
HO CH2
O CH2
P
CH3O
Peptide is cleavable with 0.5-1.0% TFA in DCM resulted in protected peptide fragments.
O
TFA
H3C C CH3
R
O
C
CO
C H
..
NH
piperidine
O
H2C
H2C
CH2 O C NH CH C NH CH C O CH2
O
O
O CH2
Wang-resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Wang resin
Type of resins for Fmoc-chemistry
Advantages:
excellent pressure stability (continuous flow synthesis) excellent swelling properties (also in water) high diffusion rates available with many types of functional groups low capacity (0.2-0.6 mmol/g), suitable for the synthesis of aggregating peptides, for on resin cyclisation and fragment condansation.
Piperidine/DMF (1:4, V/V) is added to the mark;
The mixture is shaken thoroughly and left for 25-30 min;
The resin is filtered off and the absorbance of the filtrate is measured at 301 nm (e = 7800).
AcCl or SOCl2 in toluene. Similar to ClTrt resin.
OCH3 H3CO
H2N CH
2,4-dimethoxy-benzhydryl linker NovaSyn R TGR resin
Similar to Rink Amide MBHA resin O
OCH2 C OH
OCH2-P
Rink Amide-AM and Rink Amide-MBHA resins:
OCH3
H3CO Fmoc-HN CH
OCH2-CO-Nle-R
H2N CH2
P
Aminomethyl-PS-DVB
CH3
Peptide cleavage with 90-95% TFA solution.
enantiomerisation!
Determination of loading
(Calculation of the resin capacity) 1.
10-20 mg of dried resin are weighted exactly into a 100 mL measuring flask (for a load of ca. 0.5 meq/g 20 mg is sufficient);
90% TFA solution for effective removal.
-SH (Cys)
trityl (Trt)
For selective deprotection
CH2 NH C CH3 O
acetamidomethyl (Acm)
Racemisation during the attachment of Cys derivatives to the resins
4-Alkoxybenzyl alcohol (Wang) resin:
HO CH2
O CH2
P
Attachment of the first amino acid: Fmoc-Aaa(X)-OH:DIC:DMAP (2:2:0.2 equiv to the resin OH content) in DMF, 1h at RT.
C H3 Trt group can be used if on-resin derivatization is required (glycosylation,
phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs
The final cleavage results in peptides with COOH group at the C-terminus
The resin is not available for the synthesis of peptides with a sequence on the C-terminal that is sensitive for diketopiperazine formation !
The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG) NH2 PEG
HO C H2
O O CH2 C OH
4-hydroxymethylphenoxyacetic acid linker
NovaSyn R TGA resin
Mgly =297
Rink Amide Resin: synthesis of peptides with CONH2 C-terminus
Cleavage with high concentration of TFA can lead to the break down of
OCH3
the linker
byproducts.
Use low TFA concentration and/or
trialkylsilanes in the cleavage mixture.
Peptide-resin bond can be detached
H3CO
with 5% TFA. Removal of protecting Fmoc-HN CH groups needs a separate step.
4-Hydroxymethylphenoxyacetic acid (HMPA) linker:
HO CH2
O CH2 COOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with TFA
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:
Solid phase peptide synthesis
Part II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös L. University Budapest, Hungary
Similar to Wang resin
4-carboxytrityl linker
O
NovaSyn R TGT alcohol resin
HO
C OH Before use the resin must be converted
to the chloride form by heating with
Cl Cl
The final cleavage results in peptides with COOH group at the C-terminus
Cleavage with 90-95% TFA + scavangers results in free peptides Cleavage with AcOH:MeOH(TFE):DCM (1:1:8 or 2:2:6) results in protected
HO CH2 CH3O
O (CH2)3 C OOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (ClTrt) resin:
P
Attachment of the first amino acid: